Int. J. Biosci. 2015

International Journal of Biosciences | IJB | ISSN: 2220-6655 (Print), 2222-5234 (Online) http://www.innspub.net Vol. 6, No. 6, p. 119-126, 2015

RESEARCH PAPER OPEN ACCESS

Isolation and identification of sp. VL204 associated with Pistacia atlantica leaves from Dana biosphere reserve, Jordan

Khalid Y. Al-sharafa*, Muhamad O. Al-limoun

Biological Sciences Department, Mu’tah University, Mu’tah/Al-Karak, Jordan

Key words: Ulocladium . ITS . Pistacia atlantica Desf. http://dx.doi.org/10.12692/ijb/6.6.119-126 Article published on March 29, 2015

Abstract

Endophytic isolated from Pistacia atlantica Desf. which associated with leaves necrotic spotting was identified using nuclear ribosomal DNA internal transcribed spacer regions (ITS1-5.8S rDNA-ITS2) as Ulocladium sp. VL204 a fungus member of family. * Corresponding Author: Khalid Y. Al-sharafa  [email protected]

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Introduction 1998; Hill et al., 1994; Köhl et al., 2003; Wang et al., Dana biosphere reserve is the largest reserve in 2008; Vannini and Vettraino, 2000; Zitter and Hsu Jordan had area around 292.534 km2 lays in the 1990). The objectives of this study were to provide south of Jordan. The reserve characterized by the identity of Ulocladium species isolated from diseased presence of four main vegetation communities leaves of P. atlantica Desf. from Dana biosphere according to the climate and vegetation; reserve in south of Jordan. Mediterranean (23% of reserve area), Irano-Turanian (22% of reserve area), Sudanian (48% of reserve area) Materials and methods and Saharo-Arabian (7% of reserve area). Hence, the Plant sample collection reserve native flora is exposed to different abiotic and P. atlantica Desf., was used for this study because of biotic environmental stresses. presence of necrotic spots on the plant leaves. Plant leaves samples were collected from the 4th to 22th of The genus Pistacia belongs to the family May, 2014. Anacardiaceae that comprise about 70 genera and over 600 species. A mix of Pistacia atlantica Desf. Handling of plant samples and Juniperus phoenica L. occupied around 1.17 km2 Leaves samples were kept directly on ice box after of the reserve and they form around 0.4 % of the cutting from randomly selected P. atlantica Desf. plant cover of the reserve, grown up on altitude plants. In the laboratory, samples were transferred between 1000 to 1300m above the sea level (The and stored at 4°C and processed within 24 h of royal society for the conservation of nature (RSCN)- collection. For further steps the procedure of Romero Dana biosphere reserve, 2008). et al., (2001) was used. Briefly, leaf surfaces were washed thoroughly with sterilized distilled water in Bozorgi et al., (2013) reviewed wide phytochemical order to remove the epiphytic fungal flora before and pharmacological properties from various parts of surface sterilization. The washed leaves were then Pistacia species since they utilized by people widely treated with 15% NaHOCl solution diluted with for different nutritional and medicinal proposes. P. sterilized distilled water in ratio 2:1 for 5min. surface atlantica leaves extracts showed antioxidant, sterilized leaves were cut into small pieces of 2–5 mm antimicrobial activities and antidiabetic (Benhammou squares for endophytic fungi isolation. et al., 2008; Hamdan and Afifi, 2004; Kasabri et al., 2011; Peksel, 2008). Isolation of endophytic fungi The surface-sterilized leaf segments were aseptically Ulocladium genus belongs to the family transferred to petridishes containing potato dextrose Pleosporaceae of the . Comprehensive agar (PDA) medium and incubated for 14 days at 25 systematic analysis of the Ulocladium genus using °C. three genetic loci also included related , Embellisia, and Stemphylium spp. the findings were The pure endophytic fungi strains were transferred to the taxonomic status of Ulocladium as a new PDA petridishes. monophyletic clade. In addition, to provide final resolution of this clade other more informative loci DNA extaction, PCR Amplification and DNA may provide additional support for this resolution Sequencing (Runa et al., 2009). The procedure of White et al., (1990) was used for total deoxyribonucleic acid (DNA) extraction from Ulocladium species cause leaf spots, suppress isolated fungus strain. Mycelia DNA for extraction sporulation and other diseases of different were scraped from 12-day-old PDA cultures of pure agricultural crops and plan organs (Elmer and Köhl, isolate. Followed by polymerase chain reaction (PCR)

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Int. J. Biosci. 2015 amplification of nuclear ribosomal DNA internal Primer extension sequencing was performed by transcribed spacer (ITS) regions (ITS1-5.8S rDNA- GENEWIZ, Inc. (South Plainfield, NJ) using Applied ITS2) then sequenced using primers ITS5 and ITS4 Biosystems BigDye version 3.1. Both forward and that designed also by White et al., (1990) . Primer reverse strands were sequenced. The reactions were pair ITS4 and ITS5 were used to amplify the 5.8S then run on Applied Biosystem's 3730xl DNA gene and flanking ITS1 and ITS2 regions. Analyzer.

Fig. 1. Dana reserve map with area of study. Red lined area represent the reserve area with mediterranean climate and area with blue circle is the study area.

Soil electric conductivity and soil pH characterized by semi –arid mediterranean climate Soil samples were collected using a soil auger. Four with winter temperature reached to -10 oC and sub-samples were taken from the sample area at rainfall average around 100-350mm/ year and dry different soil depth (20cm, 40cm and 60cm) and moderate summer. The western part of the reserve is mixed thoroughly to take representative soil sample characterized by dry desert climate (arid climate) with then samples dried for three days. rainfall average around 50 mm/ year (RSCN, 2008).

10g of soil sample that collected was dissolved in 25 Results ml dH2O and incubated at 25 °C with shaking at 150 Area of study rpm for 1h. The electric conductivity (EC) measured Figure 1 represent the map of Dana biosphere reserve of the soil slurry using conductivity meter 4310 with the area lined with red color characterized with (JENWAY) and the pH readings was taken by using mediterranean climate and soil and the blue circle is WTW pH 330i pH meter. the studied area that occupied with a mixture of P. atlantica Desf. and J. phoenica L. Dana reserve climate Information related to soil type, temperature and Soil of the study area characterized with pH above 8.0 rainfalls of the study area and Dana reserve were even in depth around 60 cm below the surface. Where extracted from RSCN reports (2008). Typical the soil EC was also above 300 µS/cm reaching to 420 Mediterranean soil was of the study area with most of µS/cm at depth of 60 cm (Table 1). it is calcareous soil. The eastern part of the reserve is 121 Al-sharafa and Al-limoun

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Table 1. Soil electric conductivity (EC) and pH; Data were represent means ± SD, n=3. Soil character EC (µS/cm) pH Soil depth 20cm 368±1.4 8.1±0.14

40cm 311.33±2.6 8.053±0.0094

60cm 420±0.47 8.03±0.008

Etymology and many conidia were septate were seen (Fig. 2A & Colonies grow moderately rapidly on PDA, attaining C). 45-50 mm diam in 12 d at 25oC, at first whitish, later becoming gray, with abundant dark hyphal bundles in Sequence analysis central part, reverse black (Fig. 2A). Nucleotide-nucleotide BLAST (Megablast) search using the sequence of the 549 bp amplicon against the The conidiophores varied from long, flexuous, and nr database of NCBI (www.ncbi.nlm.nih.gov) simple to short, geniculate, and branched. Conidia suggested the isolated strain was Ulocladium sp. were spherical to ellipsoidal, dark brown, verrucose VL204 with 99% significant alignments and the gene bank accession number is JF440622.1.

Table 2. Species, strains number and Sequences retrieved from GenBank accession numbers for the construction of phylogenetic tree. * indicate that unpuplished in journals. Species Strain number GenBank accession No. (ITS) Citation Alternaria petroselini EGS 09-159 AF229454.1 Pryor and Gilbertson, 2000 Alternaria smyrnii EGS 37-093 AF229456.1 Pryor and Gilbertson, 2000

Ulocladium chartarum ATCC 18044 AF229488.1 Pryor and Gilbertson, 2000

Alternaria alternata EGS 34-016 AF347031.1 Pryor and Michailides, 2002

Coniothyrium palmarum CBS 400.71 AY720708 Lennox et al., 2004

Alternaria brassicae RGT-S32 HQ674659.1 *

Ulocladium sp. VL204 JF440622.1 *

Aureobasidium melanogenum CBS 105.22 FJ150886.1 Zalar et al., 2008

Edenia gomezpompae CBS 124106 FJ839619 Crous et al., 2009

Stagonosporopsis cucurbitacearum CBS 233.52 EU167573.1 Simon et al., 2009

Leptospora rubella CPC 11006 DQ195780 Crous et al., 2006

Ulocladium chartarum U13-8 JQ585683.1 *

Ulocladium alternariae BMP 31-41-05 AF229485.1 Pryor and Gilbertson, 2000

Ulocladium consortiale U13-2 JQ585682.1 *

Phylogenetic analyses processing (neighbor joining method) (Fig. 3). The sequence analysis of the isolated fungus Ulocladium sp. VL204 was confirmed by phylogenetic Discussion tree constructed with the sequences of Ulocladium sp. Isolated Ulocladium sp. from P. atlantica Desf. leaves VL204 and similar taxa retrieved by BLAST search of Dana biosphere reserve considered the first (Table 2). The software MEGA version 4.0 (Tamura et reported study. The study was influenced by clear al., 2007) was used for the phylogenetic analyzing death of the apical part of the Pistacia trees and the

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Int. J. Biosci. 2015 necrotic spots on the leaves in particular the older pH value of calcareous soil that forms most of one. Mediterranean soil of the study area that considered moderately alkaline, however, soils with pH as high as 8.0 are consider productive and are commonly planted (Sibbett, 1995). The EC values at levels far away from the salinity soil EC values that used as indirect indicator of water content and water-soluble nutrients available for plant uptake and also without affecting on soil microorganism activity which is vital for soil processes such as respiration, residue decomposition, nitrification, and denitrification (Adviento-Borbe et al., 2006; Smith and Doran, 1996).

The phylogenetic analysis of the isolate fungus Ulocladium sp. VL204 after sequenced DNA internal transcribed spacer regions (ITS1-5.8S rDNA-ITS2) using genera of the same family indicates its very close to Alternaria brassicae strain RGT-S32 with high bootstrap, a pathogen that affect most cruciferous crops and influenced by climate with most effective in mild, wet seasons and in areas with relatively high rainfall (Humpherson-Jones and Phelps, 1989). The species grows in the vascular system and rapidly infects the entire plant (Valkonen and Koponen, 1990). However, also previous finding reported by Vannini and Vettraino, (2000) as result of wet summers, U. chartarum which is associated with high incidence of leaf spotting. However, low bootstrap was with Coniothyrium palmarum strain CBS 400.71 which is one of genera. Also the low bootstrap with other Alternaria sp.

Woudenberg et al., (2013) delineated the phylogenetic lineages within Alternaria and allied genera, and created a robust into 24 section based on nucleotide sequence data of parts of Fig. 2. A) Ulocladium sp. colony appearance on PDA the 18S nrDNA, 28S nrDNA, ITS, GAPDH, RPB2 and after 12 days incubation at 25oC. B) Micrographs of TEF1-alpha gene regions. A. brassicae was not the isolate. Flexuous conidiophores with conidia, assigned to one of the 24 Alternaria sections and is secondary conidiophore (arrow) and conidia treated as separate, single species and lineage. (magnification, 400). (C) Verrucose conidia. Septate However, these study along with others as Runa et al., is indicated by an arrow. (2009) improved that the include of other more informative loci may provide additional support for Soil pH values at different depths showed the normal this resolution and also the use of metabolites

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Int. J. Biosci. 2015 analysis (Andersen and Hollensted, 2008) to melanogenum strain CBS 105.22 sequence was distinguish these related genera which do not always chosen as out group which clear that it is not close to correlate to species-groups based upon morphological other genera by low bootstrap. characteristics. Using strain Aureobasidium

Fig. 3. The optimal Neighbor-Joining tree constructed using sequences of Pleosporales genera and that of Ulocladium sp. VL204.

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